Rat Cornea Research Articles

Effect of fluoroquinolones on the expression of matrix metalloproteinase in debrided cornea of rats

Matrix metalloproteinases (MMPs) are implicated in regenerative and healing processes in corneal injuries. Based upon reports that topical fluoroquinolones (FQs) may cause perforations during corneal healing by modulating MMPs, this study evaluated the comparative effects of commercially available FQs eye drops on the expression of MMP-2 and MMP-9 in the cornea after ethanol injury. Uniform corneal epithelial defects were created using 70% ethanol in the right eye of the rats (n = 6). The groups studied were (I) sham, (II) normal saline with benzalkonium chloride (NS-BKC), (III) norfloxacin 0.3%, (IV) ciprofloxacin 0.3%, (V) lomefloxacin 0.3%, (VI) sparfloxacin 0.3%, (VII) gatifloxacin 0.3%, and (VIII) moxifloxacin 0.5%. Each treatment was instilled six times/day up to 48 h and rats were sacrificed using excess of anesthesia. The corneas were excised to study the expression of MMP-2 and MMP-9 using gelatin zymography and real-time PCR. All the FQs significantly increased the expression of MMP-2 and MMP-9 as compared to the sham and NS-BKC-treated group. NS-BKC did not show a significant effect on MMPs expression compared to the sham group. Among the studied FQs, ciprofloxacin was observed to exhibit maximal induction of MMP-2 and MMP-9, whereas lomefloxacin exhibited an equivocal effect on both MMP-2 and MMP-9 expression. Findings of the present study demonstrate that topical application of FQs may induce the expression of MMP-2 and MMP-9 in debrided corneal epithelium and, therefore, may delay corneal wound healing. Thus, it can be concluded that selecting a FQ for ophthalmic use having minimal effect on MMPs may impact wound healing in injured or vulnerable cornea.

Changes of the thiol levels in the corneas of the diabetic rats: effect of carnosine, aspirin and a combination eye drops.

To investigate the effect of carnosine (Car), aspirin (Asp) and a combination of Car and Asp eye drops on the change of the thiol contents from glutathione (GSH) and protein in the corneas of the diabetic rats. All the animals were randomly divided into five groups. The normal control group received injections of vehicle only. Diabetes was induced by injection of streptozotocin (STZ) in the Sprague-Dawley (SD) rats. The untreated group rats received only the vehicle solution (the placebo). One treated group rats were treated by instillation of one drop of 10g/L Car eye drops, another were treated by 0.5g/L Asp eye drops and the last group were treated alternately by Car 10g/L and Asp 0.5g/L eye drops for a period of 8 weeks. At the end of 8 weeks, the animals were killed and the thiols contents in the corneas were investigated. About 15.6% of the rats (blood glucose measured <14mmol/L) were rejected. In the corneas, the levels of thiols were declined in the untreated, Asp-treated group and combination-treated group, but they went up in Car-treated group. The levels of thiols in the Car-treated groups were much higher than that in the untreated group, and there was statistically significant difference between them (P<0.05). The results indicated that diabetes decreases the levels of thiols (from GSH and proteins) in the cornea. The Car eye drops in our study may protect the cornea against the oxidative damage caused by diabetes. And the combination eye drops also may have a certain protection for the diabetic corneas.

Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury.

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α-stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.

Increased gene expression of the apoptosis‐related molecule Fas and the inducible nitric oxide synthase (iNOS) after excimer laser ablation in rat corneas

Abstract Purpose The aim of this study was to analyse the gene expression of the apoptosis‐related molecule Fas in relation to the production and accumulation of nitric oxides 1, 12 and 24h after excimer laser keratectomy in rat corneas. Methods Wistar rats with excimer corneal ablations (Chiron laser, 66‐68 µm/pulse, 1606 pulses, diameter 4‐7.6 mm, and depth in cornea 50µm) were killed 1, 12 and 24h after treatment. Fas mRNA in situ hybridizations were performed in the analysed corneas by using a rat‐Fas specific gene probe. The generation and further accumulation of reactive nitrogen intermediates (RNI) due to the activity of the inducible nitric oxide synthase (iNOS) in corneal cells were further evaluated by systematic mRNA in situ hybridizations with a specific gene probe for iNOS. Results One hour after treament, the Fas gene expression was higher than in untreated controls, but clearly lower than 12h after treatment. Whereas 24h after treatment, the expression remained higher than in the controls, slight decreases were observed when compared to the corresponding levels 12h after treatment. A similar expression pattern was observed for the accumulation of RNI in plasma. This effect was further confirmed in corneal cells by the detection of similar iNOS hybridization patterns in the corresponding corneas. Conclusion After excimer laser keratectomy, the Fas gene expression was increased, especially 12h after treatment. These increases were found to be related to the accumulation of RNI in plasma and to the expression of iNOS in corneal cells. The expression of all these three considered markers together may play a relevant role in wound healing after laser treatment.

Cellular genic therapy: evaluation of mesenchymal stem cells engraftment transduced by an Ecdysone inductible lentiviral vector expressing luciferase gene

Abstract Purpose Gene therapy might offer an opportunity to improve new treatments in ophthalmology. Several studies, using limbal stem cells or mesenchymal stem cells (MCSs), have confirmed the usefulness of these cells to repair epithelial and stromal corneal tissue. Methods We designed a strategy aimed at expressing Luciferase gene. We have tested the viability of mesenchymal stem cells modified by a lentiviral vector delivery system. To allow an adaptative treatment, the target genes would be transcriptionally controlled by the use of an Ecdysone inductible promoter. Results Construction of a lentiviral plasmid with the transgene (luciferase) and inductible promoter: Ecdysone by recombination. Construction of a lentiviral plasmid with the RXR gene and VgEcR gene and a constitutive enhancer. Transfection of 293 FT cells for lentiviral production. MSCs Transduction by “V/R” lentivirus and by “E/Transgene” lentirus. We have tested induction of Ecdysone system and transcription of the transgene (luciferase) in MSCs adding Ponasterone. Conclusion The expression of luciferase, using conditionnal expression vectors, could be applied after an engraftment of mesenchymal stem cells for pathological wound healing in Sprague‐Dawley rat cornea.

Histopathological effects of maternal hair dye use on the cornea

The aim of this study is to investigate and compare the histopathological effects of hair dye additives, 2-amino-5-nitrophenol (2A5NP) and 2-nitro-p-phenylendiamin (2NPPD) on cornea of neonates from pregnant rats that have been administered these additives subcutaneously. The study included 90 neonates of 26 nulligravida wistar-albino rats among which ten were given 100 mg/kg/day 2A5NP (Group I), ten rats received 150 mg/kg/day 2NPPD (Group II) and control rats received saline (Group III) injections subcutaneously between 7th and 15th gestational days. No sign of toxicity was observed during the treatment and there was no gross abnormality in both the study and control groups. Histopathological changes of cornea were seen in 22 of 30 newborn rats in Group I (73.4%), in 23 of 30 rats in Group II (76.7%) and only 5 of 30 rats in the control saline injected Group III (16.7%). Histopathological effect of the two additives were statistically significant when compared to the control group (Chi-square:27.63, p = 0.0001), but there was no difference between the effects of 2A5NP and 2NPPD additives on cornea (Chi-square:0.089, p = 0.766). The present experimental study on rats confirmed the histopathological effect of 2A5NP and 2NPPD on cornea beyond doubt. In the light of which, we can speculate that maternal exposure of hair dyes during pregnancy has some teratogenic effects on newborn rat cornea. Key words: 2-Amino-5-nitrophenol, 2-nitro-p-phenylendiamin, cornea, neonatal rats.

Melatonin promotes angiogenesis during protection and healing of indomethacin-induced gastric ulcer: role of matrix metaloproteinase-2

Matrix metalloproteinase (MMP)-2 is considered as a crucial regulator of angiogenesis, a process of new blood vessel formation. We reported previously that melatonin (N-acetyl-5-methoxy tryptamine), an antioxidant and anti-inflammatory agent, prevents indomethacin-induced gastric ulcers. Herein, we investigated the effect of melatonin on MMP-2-mediated angiogenesis during gastroprotection. Angiogenic properties of melatonin were tested in both rat corneal micropocket assay and in mouse model of indomethacin-induced gastric lesions. Melatonin augmented angiogenesis that was associated with amelioration of MMP-2 expression and activity and, upregulation of vascular endothelial growth factor (VEGF) in rat cornea. Melatonin prevented gastric lesions by promoting angiogenesis via upregulation of VEGF followed by over-expression of MMP-2. Similarly, healing of gastric lesions was associated with early expression of VEGF followed by MMP-2. In addition, upregulation of MMP-2 was parallel to MMP-14 and inverse to tissue inhibitor of metalloprotease (TIMP)-2 expression during gastroprotection. Our data demonstrated that melatonin exerts angiogenesis through MMP-2 and VEGF over-expression during protection and healing of gastric ulcers. This study highlights for the first time a phase-associated regulation of MMP-2 activity in gastric mucosa and an angiogenic action of melatonin to rescue indomethacin-induced gastropathy.

Inhibition of corneal neovascularization after alkali burn: comparison of different doses of bevacizumab in monotherapy or associated with dexamethasone

To compare the effects of different doses of bevacizumab with both saline and dexamethasone on inflammatory angiogenesis in the rat cornea induced by small chemical lesions. Corneal chemical cauterization was performed on 24 rats. Animals were divided randomly into six groups and received a daily subconjunctival injection for 7 days of: balanced salt solution 0.1 mL or dexamethasone phosphate 4 mg/day or bevacizumab 2.5 mg/day, 3.75 mg/day, 5.0 mg/day or bevacizumab 5.0 mg/day + dexamethasone phosphate 4 mg/day. Clinical examination under slitlamp was performed daily for 7 days to evaluate corneal opacity and vessel size evolution. Computer assisted quantitative image analysis was used to measure the total corneal area covered by neovascularization. At final examination, the dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups showed a significant lowering in corneal opacity score as compared with control (P = 0.024, P = 0.006 and P = 0.013, respectively). Also, a significant reduction on new vessels size score was observed. Surface of corneal neovascularization was significantly reduced in dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups compared with control (P =0.045, P = 0.047 and P = 0.044, respectively). Our study demonstrates the ability of a 5.0 mg/day bevacizumab subconjunctival injection, in monotherapy or associated with dexamethasone, to cause a short-term involution of corneal neovascularization after corneal alkali burn. Combination of both of these treatments may have advantages to monotherapy approaches.

Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist

Analgesics currently available for the treatment of pain following ophthalmic surgery or injury are limited by transient effectiveness and undesirable or adverse side effects. The cornea is primarily innervated by small-diameter C-fiber sensory neurons expressing TRPV1 (transient receptor potential channel, subfamily V, member 1), a sodium/calcium cation channel expressed abundantly by nociceptive neurons and consequently a target for pain control. Resiniferatoxin (RTX), a potent TRPV1 agonist, produces transient analgesia when injected peripherally by inactivating TRPV1-expressing nerve terminals through excessive calcium influx. The aim of the present study was to evaluate topical RTX as a corneal analgesic. In rat cornea, a single application of RTX dose dependently eliminated or reduced the capsaicin eye wipe response for 3–5 days, with normal nociceptive responses returning by 5–7 days. RTX alone produced a brief but intense noxious response, similar to capsaicin, necessitating pretreatment of the cornea with a local anesthetic. Topical lidocaine, applied prior to RTX, blocks acute nociceptive responses to RTX without impairing the subsequent analgesic effect. Importantly, RTX analgesia (a) did not impair epithelial wound healing, (b) left the blink reflex intact and (c) occurred without detectable histological damage to the cornea. Immunohistochemistry showed that loss of CGRP immunoreactivity, a surrogate marker for TRPV1-expressing fibers, extended at least to the corneal-scleral boundary and displayed a progressive return, coincident with the return of capsaicin sensitivity. These data suggest that RTX may be a safe and effective treatment for post-operative or post-injury ophthalmic pain.

The Inhibitory Effects of Bevacizumab Eye Drops on NGF Expression and Corneal Wound Healing in Rats

To investigate the effect of bevacizumab eye drops on corneal epithelial wound healing in rats. One hundred twenty Sprague-Dawley male rat corneas were divided into two groups and de-epithelized with a microblade. The bevacizumab group was treated with 5% bevacizumab and antibiotic 0.5% levofloxacin eye drops four times daily and the control group with antibiotic eye drops only. Corneal wound healing was evaluated by fluorescein staining at initial wounding and 24, 48, and 72 hours after epithelial debridement. Nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) proteins were measured in rat corneas by ELISA. Immunofluorescent staining for NGF and VEGF was performed in rat corneas. NGF mRNA and VEGF mRNA was measured in rat corneas by real-time PCR. The corneal wound healing rate was decreased in the bevacizumab group compared with that in the control group. Twenty-four, 48, and 72 hours after epithelial debridement, corneal NGF and VEGF proteins in the bevacizumab group were lower than that in the control group (P<0.05). Immunofluorescent staining showed that NGF and VEGF expression was stronger in the control group than in the bevacizumab group. NGF mRNA and VEGF mRNA levels in the bevacizumab group were also lower than in the control group (P<0.05). After corneal epithelial damage, VEGF and NGF increased normally in the rat corneas. In contrast, when VEGF was inhibited by bevacizumab eye drops, the wound healing rate was decreased, and NGF was downregulated. Bevacizumab eye drops have an inhibitory effect on corneal wound healing in rats.

Effects of Ultraviolet B Radiation on the Cornea of Adult Male Albino Rats and the Possible Role of Lornoxicam

Introduction: Ultraviolet Radiation (UVR) represents a significant environmental and occupational hazard to human beings especially after the ozone hole. Lornoxicam is a strong non-steroidal anti-inflammatory drug (NSAID) with few side effects. Aim of the Work: This work aimed to clarify effects of UVBR on the cornea of adult male albino rats and the possible role of lornoxicam. Materials and Methods: Thirty adult male albino rats were used. They were divided into three main groups: Control; Lornoxicam treated and UV irradiated which was further subdivided into 3 subgroups. On sacrifice, cornea was processed for histological, immunohistochemical and morphometrical studies. Results: Cornea of UV irradiated rats revealed epithelial thinning with cellular degeneration, exfoliation and metaplasia. Bowman's membrane was focally interrupted or absent. Stroma showed irregularly arranged widely separated collagen fibers, degenerated few keratocytes with neovascularization, interstitial hemorrhage and cellular infiltration. Ultrastructurally, epithelial cells showed nuclear membrane irregularities, chromatin condensation, mitochondrial degeneration and loss of rER. Immunohistochemically, epithelial cells and keratocytes showed marked expression of caspase-3 protein in their cytoplasm. Morphometrically, there was a highly significant decrease in epithelial thickness, area % of stroma and number of keratocytes. Rats left to recover without treatment showed mild improvement, while treatment of irradiated rats with lornoxicam for one week after exposure showed nearly normal histological, ultrastructural, and immunohistochemical pictures. Conclusion: Lornoxicam (a cheap, available tolerable NSAID) could ameliorate UVB induced corneal changes. So it could be considered as a curative measure of the cornea against UVB.

Expression of AQP-1 in rat cornea neovascularization after alkali burn

Background Corneal alkali burn is a common cause of corneal neovascularization(CNV).Researches demonstrated that aquaporin 1(AQP1) plays key role in neovascularization of tumor tissue.So it is speculated that AQP1 participates in pathogenesis and development of CNV.ObjectivePresent study aims to investigate the expression of aquaporin 1(AQP1) in cornea after alkali burn and explore its role in corneal neovascularization.MethodsCornea neovascularization (CNV) models were established by putting a filter paper (the size of 3 mm in diameter immersed 1mol/L NaOH solution) on cornea for 20 seconds in the left eyes of 25 adult Sprague Dawley rats.The fellow eyes were treated using the filter paper with normal saline solution as normal control group.Corneal new blood vessel was evaluated under the slim lamp biomicroscopy once per day,and area of new vessel was calculated.The animals were sacrificed and corneal samples were obtained in day 1,4,7,14,21 after corneal alkali burn.The expression of AQP1 and VEGF in corneas were detected by immunochemistry,and the relative expression level of AQP1 mRNA in rat cornea was determined using RT-PCR.ResultsThe corneal neovascularization grew at the second day and reached the largest area at the fourteenth day after corneal alkali burn.The expression of VEGF protein (gray value) in corneal sample was significantly elevated from the first day through the fourteenth day after corneal alkali burn in comparison with control group(t_(1d)=12.410,t_(4d)=29.428,t_(7d)=16.030,t_(14d)=13.321,P＜0.05),and then the expression level declined till twenty-one day without obvious difference in comparison with control group(t_(21d)=1.587,P＞0.05).The expression of AQP1 protein showed the same tendency with that of VEGF protein(t_(1d)=7.623,t_(4d)=15.293,t_(7d)=18.291,t_(14d)=14.483,t_(21d)=6.381,P＜0.05).The dynamic change expressions of AQP1 and VEGF showed a significant positive correlation(r=0.834,P＜0.05).The expression of AQP1 mRNA (A value)was weaker in control group but considerably increased after alkali burn with the peak value in the seventh day,showing a significantly difference between day 1,4,7,14 and control group(t_(1d)=3.491,t_(4d)=10.690,t_(7d)=12.936,t_(14d)=10.767,t_(21d)=8.594,P＜0.05).ConclusionThe level of AQP1 expression is markedly related to inflammatory CNV in rat cornea after alkali burn. Key words: aquaporin1; VEGF; cornea alkali burn; corneal neovascularization

Endovascular Treatment Increases but Gamma Knife Radiosurgery Decreases Angiogenic Activity of Arteriovenous Malformations

To compare the angiogenic potentials of embolized, gamma knife-treated or untreated cerebral arteriovenous malformations (AVMs), using a rat cornea angiogenesis model. Tissue samples from cerebral AVM patients who were either untreated or had previously been treated with embolization or gamma knife radiosurgery and who had undergone operations for hemorrhage at the Neurosurgery Department or the Neurological Sciences Institute of Marmara University were used. For the macroscopic evaluation of angiogenesis, tissue samples were inoculated in a micropocket created on the rat eye, and the level of angiogenic activity was graded macroscopically for 15 days, with glioblastoma multiforme and normal brain artery tissues serving as positive and negative controls, respectively. For the other part of the experiment, eyes of another set of rats were inoculated with the study samples only using the same cornea angiogenesis model, in which microvessel count and vascular endothelial growth factor assessment was done at days 3, 7, 11, and 15. Based on our macroscopic findings in the cornea angiogenesis model, embolized AVMs exhibited the highest angiogenic activity, followed by untreated AVMs and gamma knife-treated AVMs. Evaluations of vascular endothelial growth factor expression and microvessel counts showed a similar relation among the 3 tissue groups with regard to the level of angiogenic activity, supporting the results of macroscopic examinations. This study, for the first time, provides experimental semiquantitative data to compare the angiogenic potentials of embolized and gamma knife-treated AVM tissues. Embolization may increase angiogenic activity, and gamma knife radiosurgery may decrease it when compared with activity in previously untreated AVMs. These data can be useful to understand why recurrence of AVMs after angiographically demonstrated endovascular occlusion is common but after gamma knife occlusion is rare.

Zinc-finger transcriptional factor Sall1 induces angiogenesis by activation of the gene for VEGF-A

Zinc-finger transcriptional factor Sall1 modulates gene expression and regulates organogenesis, including kidney development. Angiogenesis induced by vascular endothelial growth factor (VEGF) is also required for organogenesis. We investigated whether Sall1 induces angiogenesis through VEGF gene activation. Sall1 gene transfer induced marked neovascularization in rat cornea and in mouse embryoid bodies (EBs). The neovascularization in EBs was abolished by co-administration of anti-VEGF antibody. Sall1 gene transfer in Swiss 3T3 cells significantly increased the expression of VEGF-A mRNA but did not markedly increase the expression of fibroblast growth factor-2, epidermal growth factor, hepatocyte growth factor and ETS-1 mRNA. Sall1 gene transfer significantly increased VEGF-A protein levels in conditioned medium from cultured fibroblasts. Sall1 gene transfer significantly increased VEGF-A promoter activity in HEK293T cells as compared with cells transfected with mock vector or truncated Sall1. These results suggest that Sall1 induces angiogenesis by stimulating VEGF-A promoter activity.

Role of epidermal growth factor receptor (EGFR) in corneal remodelling in diabetes

This study examined the role of epidermal growth factor receptor (EGFR) signalling on the organization and remodelling of collagen fibrils (CFs) and proteoglycans (PGs) in the stroma of diabetic rat cornea. Diabetes was induced in female Wistar rats (n = 5) by streptozotocin (STZ) injection (55 mg/kg). Treatment with a selective inhibitor of EGFR tyrosine kinase, AG1478, was started on the same day as the induction of diabetes and administered every other day for 4 weeks. Corneas were fixed in 4% paraformaldehyde at 4 degrees to allow for analysis of CF diameters and in 2.5% glutaraldehyde in sodium acetate buffer containing cuprolinic blue to enable the study of PG distribution. AnalySIS soft imaging software was used to analyse CFs and PGs. Epithelial thickness, and median diameter and area fraction of CF in corneal stroma were decreased in diabetic rat cornea compared with normal cornea (p < 0.001), whereas the median PG area and area fractions were significantly increased (p < 0.001). Treatment with AG1478, although it had no action on normal cornea, prevented these diameter and area fraction changes in CFs and PGs. The cornea of AG1478-treated diabetic rats showed a slight increase in CF diameter and area fraction and a decreased number density. These data show that the distribution of corneal stroma CFs and PGs was altered after 4 weeks of diabetes and that, furthermore, treatment with an EGFR signalling inhibitor normalized these abnormalities. The data suggest that EGFR plays an important role in the development of diabetes-induced corneal remodelling.

The effects of steriod on expression of TLR2 in rat corneas after penetrating keratoplasty

目的 检测大鼠角膜组织TLR2 mRNA的表达,研究糖皮质激素对穿透角膜移植术后角膜TLR2表达的影响及对术后排斥反应的作用.方法 实验动物分正常对照组(N)、同种同体角膜移植组(A)、同种异体角膜移植组(B)和同种异体角膜移植激素(典必殊滴眼液点术眼,每日2次)抗排斥组(C),A、B、C 3组大鼠给予穿透角膜移植术,术后裂隙灯下观察角膜透明度、新生血管并用排斥反应指数评价;分别于术后第5、7、9天取材,行组织病理学检查和实时荧光定量PCR检测,动态观察角膜组织中TLR2 mRNA的表达.结果 术后随时间变化角膜植片均出现不同程度的水肿、混浊、新生血管生长.B组大鼠平均在穿透角膜移植术后7d发生排斥反应并获病理学支持;A组和C组大鼠术后排斥反应指数计分未达到诊断标准.术后3组手术干预组角膜TLR2 mRNA的表达均增加,同种异体角膜移植组角膜TLR2 mRNA的表达较同种同体角膜移植组显著增加,激素抗排斥组角膜TLR2 mRNA表达明显降低(P＜0.05).结论 糖皮质激素可能通过抑制角膜TLR2的表达及其介导的信号转导,抑制排斥反应,对移植物起保护作用。

Ultrapure chitosan oligomers as carriers for corneal gene transfer

NOVAFECT chitosans are ultrapure chitosan oligomers that were recently marketed as carriers for non-viral gene therapy. There are no reports on systematic design and improvement of formulations based on NOVAFECT chitosans for gene delivery. Therefore, we have designed and characterized chitosan-DNA nanoparticles based on NOVAFECT. We found that the size of oligomeric chitosan-DNA nanoparticles is small, ≤98.2 ± 4.4 nm. Zeta potential measurements of oligomeric chitosan-DNA nanoparticles exhibited a strong positive charge ≥44.1 ± 3.5 millivolt. In vitro transfection studies demonstrated the ability of oligomeric chitosan-DNA nanoparticles to effectively transfect COS-7 cells. In rat corneas, injection of a select formulation of oligomeric chitosan-DNA nanoparticles into the stroma showed that (a) luciferase gene expression was 5.4 times greater than following administration of polyethylenimine-DNA nanoparticles; and (b) the cells that express the transgene, green fluorescent protein, were keratocytes (corneal fibroblasts). This study lays the foundation for evaluating oligomeric chitosan-DNA nanoparticles as pharmaceuticals for corneal gene therapy, a promising approach for the treatment of acquired and inherited corneal diseases that otherwise lead to blindness. Oligomeric chitosan-DNA nanoparticles can also be evaluated for the treatment of ocular diseases outside of the cornea, and for various additional gene therapy applications.

The Effect of Lyophilization on Graft Acceptance in Experimental Xenotransplantation Using Porcine Cornea

The immunogenicity of lyophilized porcine cornea is unknown. The purpose of this study was to examine the possibility of using lyophilized porcine cornea as a substrate for ocular surface reconstruction. A porcine cornea stromal button was freeze-dried and vacuum-packed. Lyophilized and fresh porcine corneas were examined histologically, and then implanted into intrastromal pockets in live rat corneas. Cytokine concentrations in plasma and protein extracts from the corneal buttons of rats were measured using the fluorokine multianalyte profiling assay, and histologic examination was performed. Immunoreactivity to the alpha-gal epitope was not found in lyophilized porcine corneas, whereas it was found in several keratocytes in fresh porcine corneas. The median survival time of rat corneas receiving lyophilized porcine transplants was 28.0 days, significantly longer than the 14.0-day survival of rat corneas that received fresh porcine transplants (P < 0.05). CD45RO(+) and CD68(+) cells were observed in rejected corneas, and interleukin-2 and interferon-gamma were elevated in rat plasma and corneal tissue. The lyophilized porcine corneal stroma, which is devoid of alpha-gal epitope, is less antigenic, and may be a useful biomaterial for ocular surface reconstruction and corneal collagen supplementation.

Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy

To determine whether in vivo confocal microscopy (IVCM) of the cornea can be used for the label-free detection and monitoring of lymph vessels in live corneas. Parallel corneal hemangiogenesis and lymphangiogenesis was induced by the placement of a single suture in one cornea of male Wistar rats. Fourteen days after suture placement and under general anesthesia, laser-scanning IVCM was performed in the vascularized region. Corneas were subsequently excised for flat-mount double immunofluorescence with a pan-endothelial marker (PECAM-1/CD31) and a lymphatic endothelial specific marker (LYVE-1). Using the suture area and prominent blood vessels as points of reference, the identical microscopic region was located in both fluorescent and archived in vivo images. Additionally, vessel diameter, lumen contrast, and cell diameter and velocity within vessels were quantified from in vivo images. Comparison of identical corneal regions in fluorescence and in vivo revealed prominent CD31(+)/LYVE-1(3+) lymph vessels that were visible in vivo. In vivo, corneal lymph vessels were located in the vascularized area in the same focal plane as blood vessels but had a darker lumen (P < 0.001) sparsely populated by highly reflective cells with diameters similar to those of leukocytes in blood vessels (P = 0.61). Cell velocity in lymph vessels was significantly reduced compared with blood particle velocity (P < 0.001). Morphologic characteristics enabled subsequent identification of corneal lymphatics in live, vascularized rat corneas before immunofluorescence labeling. IVCM enabled the nondestructive, label-free, in vivo detection of corneal lymphatics. IVCM provides the possibility of observing lymphatic activity in the same live corneas longitudinally and, as a clinical instrument, of monitoring corneal lymphatics in live human subjects.

Small interference RNA targeting vascular endothelial growth factor inhibits rat corneal neovascularization

To test the effects of VEGF-siRNA-transfected corneal epithelium on corneal neovascularization (CNV). It was an experimental study. Cultured rat corneal epithelial cells and keratocytes were transfected with synthesize VEGF siRNA by lipofectamine 2000. The level of VEGF mRNA was analyzed by real time PCR, and the protein levels were determined by enzyme-linked immunosorbent assay (ELISA). CNV was induced by cauterization with 1 mol/L sodium hydroxide in rat corneas. The VEGF-siRNA-transfected-corneal epithelium cells were transplanted to the CNV lesions. Immediately after transplantation, the VEGF-siRNA combined with lipofectamine 2000 were directly transfected rat cornea through injecting into the anterior chamber. After surgery, the surface areas occupied by new vessels were measured, and VEGF protein was localized by immunohistochemistry. The levels of VEGF expression at both mRNA and protein in the VEGF-siRNA transfected corneal epithelial cells and keratocytes were significantly lower than those of control cells. VEGF siRNA could inhibit the expression of VEGF mRNA in corneal epithelial cells and keratocytes to 57% - 85% and 59% - 78%, respectively. The VEGF-siRNA-transfected-corneal epithelium transplantation significantly decreased the surface areas occupied by new vessels. VEGF expression level in interference groups was lower than that in the control group. The development of CNV is markedly suppressed by VEGF-siRNA transfection in vivo.